A Versatile Approach to Attachment of Triarylmethyl Labels to DNA for

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A Versatile Approach to Attachment of Triarylmethyl Labels to DNA for Nanoscale Structural EPR Studies at Physiological Temperatures Georgiy Yu Shevelev, Evgeny L. Gulyak, Alexander Anatolyevich Lomzov, Andrey A. Kuzhelev, Olesya A. Krumkacheva, Maxim S. Kupryushkin, Victor M. Tormyshev, Matvey V. Fedin, Elena G. Bagryanskaya, and Dmitrii V. Pyshnyi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b10689 • Publication Date (Web): 05 Dec 2017 Downloaded from http://pubs.acs.org on December 6, 2017

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The Journal of Physical Chemistry

A Versatile Approach to Attachment of Triarylmethyl Labels to DNA for Nanoscale Structural EPR Studies at Physiological Temperatures Georgiy Yu. Shevelev,†,‡ Evgeny L. Gulyak,‡,† Alexander A. Lomzov,†,‡ Andrey A. Kuzhelev,‡,# Olesya A. Krumkacheva,§,‡ Maxim S. Kupryushkin,† Victor M. Tormyshev,#,‡ Matvey V. Fedin, §,‡,*

Elena G. Bagryanskaya#,§,‡,* and Dmitrii V. Pyshnyi†,‡,*

† Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 630090, Russia ‡ Novosibirsk State University, Novosibirsk 630090, Russia § International Tomography Center SB RAS, Novosibirsk 630090, Russia # N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Novosibirsk 630090, Russia KEYWORDS SPECTROSCOPY, ELECTRON DOUBLE-RESONANCE, PELDOR, ECHO, ELECTRONSPIN RELAXATION, SITE-DIRECTED SPIN LABELING, TRIARYLMETHYL RADICALS,

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PROBES, DISTANCE MEASUREMENTS, NUCLEIC-ACIDS, ROOM-TEMPERATURE, TREHALOSE

ABSTRACT

Triarylmethyl (trityl, TAM) radicals are a promising class of spin labels for nanometer-scale distance measurements in biomolecules at physiological temperatures. However, to date existing approaches for site-directed TAM labeling of DNA have been limited to label attachment at the termini of oligonucleotide, thus hindering a majority of demanded applications. Herein we report a new versatile strategy for TAM attachment at arbitrary sites of nucleic acids. It utilizes an achiral non-nucleoside phosphoramidite monomer for automated solid-phase synthesis of oligonucleotides, which are then post-synthetically functionalized with TAM. We demonstrate a synthesis of a set of oligonucleotide complexes that are TAM-labeled at internal or terminal sites, as well as the possibility of interspin distance measurements up to ~5-6 nm at 298 K using Double Quantum Coherence (DQC) Electron Paramagnetic Resonance (EPR). Implementation of the developed approach strongly broadens the scope of nucleic acids and nucleoprotein complexes available for nanoscale structural EPR studies at room temperatures.

1. INTRODUCTION Electron paramagnetic resonance (EPR) in combination with Site-Directed Spin Labeling (SDSL) is extensively used for nanoscale distance measurements and structural investigations of biological systems.1–10 As a rule, two paramagnetic moieties are incorporated into the biomolecule and dipolar interaction is measured between them using Double Electron-Electron


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Resonance (DEER or PELDOR),11–13 Double Quantum Coherence (DQC)9,14 or other Pulse Dipolar EPR Spectroscopy (PDS) techniques.15 Until recently, the majority of EPR distance measurements were carried out using pairs of stable nitroxide radicals at temperatures ~50-80 K.16,17 To enhance the information obtained from PDS experiments, pairs of different spin labels (so-called orthogonal labeling) are being increasingly used.

18–20

Another recent trend involves application of triarylmethyl (TAM)

radicals – a new promising type of spin labels with a number of improved properties, such as much narrower EPR line compared to nitroxides,21 higher phase memory time Tm in aqueous solutions,22,23 high stability in various biological reducing media including cells.24,25,26 It might also be beneficial to use TAMs as orthogonal labels in combination with other labels.27,28 The advanced properties of TAMs boost their applications to various studies in structural biology. In particular, TAMs have been successfully used for distance measurements at cryogenic near-physiological29 and true physiological30 temperatures.31,32 Several synthetic approaches for incorporation of TAM labels into the structure of proteins have been reported.27– 29

At the same time, there is a desperate need in versatile strategies for spin-labeling of nucleic

acids using TAMs. To date, only one reliable method for TAM labeling of DNA at 5' termini has been described.30,33 Certainly, this approach is not versatile and, in particular, it does not allow incorporation of TAM labels in arbitrary positions of DNA. Herein we propose a new method of TAM labeling at internal and terminal sites of oligonucleotides. It employs an achiral non-nucleoside phosphoramidite monomer for automated solid-phase synthesis.34 This non-nucleotide insertion contains an amino group protected with a trifluoroacetyl group. The use of this non-nucleoside monomer allows one to synthesize oligonucleotides that are TAM-labeled at 3' and 5' termini, as well as at arbitrary internal sites.


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2. EXPERIMENTAL SECTION 2.1. Synthesis of the non-nucleoside phosphoramidite monomer for automated solid-phase oligonucleotide synthesis. N-(2-Hydroxyethyl)morpholine-2,3-dione (Scheme 1, compound 1) was synthesized and characterized according to a previously published method.34 N-(2-(4,4-Dimethoxytrityloxy)ethyl)morpholine-2,3-dione (2). Compound 1 (scheme 1) (2 g, 12.72 mmol) was dried by coevaporation with anhydrous pyridine (20 mL) and dissolved in anhydrous pyridine (30 mL). 4,4′-Dimetoxytrityl chloride (DMTrCl) (3.9 g, 11.42 mmol) was added to the solution. The reaction mixture was stirred at ambient temperature for 3 h, evaporated to an oil, dissolved in 100 mL of CH2Cl2 and washed with aqueous solution of 0.3 M KH2PO4 (3 × 150 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to an oil. Compound 2 was obtained with the yield of 91% and applied to the next step without purification. N-(Trans-4-aminocyclohexyl)-N-(2-(4,4′′-dimethoxytrityloxy)ethyl)-N′′-(2hydroxyethyl) oxalamide (3). Compound 2 (scheme 1) (1.62 g, 3.53 mmol) was dried by coevaporation with anhydrous pyridine (2 × 10 mL) and dissolved in 20 mL of anhydrous pyridine. Trans-1,4-diaminocyclohexane (0.53 g, 4.6 mmol) was added to the solution. The reaction mixture was stirred at ambient temperature for 24 h, concentrated in vacuo, dissolved in 200 mL of CH2Cl2, and washed with a saturated NaCl solution containing 5% NaHCO3 (3 × 150 mL). The organic layer was dried over anhydrous Na2SO4, evaporated to an oil, dissolved in 5 mL of toluene and subjected to chromatography on a silica gel column, eluted with a step gradient of CH2Cl2:Et3N 99:1 (v/v) to EtOH:Et3N 99:1 (v/v). Appropriate fractions were pooled and evaporated to dryness, yielding 0.63 g of 3 (31%).


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N-(Trans-4-(2,2,2-trifluoroacetamido)cyclohexyl)-N-(2-(4,4dimethoxytrityloxy)ethyl)-N-(2-hydroxyethyl)oxalamide (4). Compound 3 (scheme 1) (0.63 g, 1.1 mmol) was dried in vacuo over P2O5 and dissolved in 10 mL of anhydrous pyridine. Ethyl trifluoroacetate (0.17 mL, 1.4 mmol) and triethylamine (0.225 mL, 1.6 mmol) were added. The reaction mixture was stirred at ambient temperature for 3 h, concentrated in vacuo, dissolved in 100 mL of CH2Cl2, and washed with aqueous solution of 0.3 M KH2PO4 (3 × 150 mL). The organic layer was dried over anhydrous Na2SO4, evaporated to dryness, dissolved in 5 mL of toluene and subjected to chromatography on a silica gel column, eluted with a step gradient of CH2Cl2:Et3N 100:1 (v/v) to CH2Cl2:acetone:Et3N 30:70:1 (v/v/v). Appropriate fractions were pooled and evaporated to dryness, yielding 0.389 g of 4 (53%). N-(Trans-4-(2,2,2-trifuloroacetamido)cyclohexyl)-N′′-(2-(4,4′′dimethoxytrityloxy)ethyl)-N′′-(2-((2-cyanoethoxy)-N,Ndiisopropylaminophosphinyloxy)ethyl)oxalamide (5). Compound 4 (Scheme 1) (0.389 g, 0.58 mmol) was dried in vacuo over P2O5 and dissolved in 2 mL of anhydrous MeCN. 5-(Ethylthio)1H-tetrazole (0.151 g, 1.16 mmol), DIEA (0.47 mL, 2.67 mmol), and 2-cyanoethyl-N,N,N′,N′tetraisopropylphosphordiamidite (0.38 mL, 1.16 mmol) were dissolved in 2 mL of anhydrous MeCN and stirred at ambient temperature for 30 min. The solution of 4 was added dropwise to the mixture during 2 min. The reaction mixture was stirred at ambient temperature for 1 h, then distilled water was added (0.010 mL, 0.56 mmol), and the mixture was stirred for another 5 min. The mixture was evaporated to 1⁄4 of its volume, diluted with 100 mL of CH2Cl2, and washed with aqueous solution of 0.3 M KH2PO4 (3 × 150 mL). The organic layer was dried over anhydrous Na2SO4, evaporated to an oil, dissolved in 5 mL of toluene, and chromatographed on a silica gel column, eluted with a step gradient of toluene:Et3N 100:1 (v/v) to toluene:ethyl


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acetate (EtOAc):Et3N 20:80:1 (v/v/v). Appropriate fractions were pooled, evaporated to dryness and dried in vacuo over P2O5. Yield of 5 was 0.383 g (75%). Rf = 0.5 (toluene – EtOAc, 1:1 v/v). The details of NMR data for synthesized compounds 2 - 5 are given in SI.

2.2. Oligonucleotide synthesis Oligonucleotides (Table 1) were synthesized on an automated DNA synthesizer ASM700 (Biosset, Russia) using High Load Controlled Pore Glass (CPG) support and standard nucleoside phosphoramidites (Glen Research, USA) at 0.05 M in anhydrous acetonitrile (MeCN). Non-nucleoside phosphoramidite 5 (Scheme S2) was employed at 0.1 M in anhydrous MeCN. The time for the condensation step with the modified monomer was increased from 1 to 15 min as compared to the standard protocol. Oligonucleotides were synthesized with a 5′dimetoxytrityl (DMTr) group on. The oligonucleotides were cleaved from the solid support and deprotected with concentrated aqueous ammonia at 56°С for 8 h. Table 1. Sequence of the synthesized oligonucleotides. [X] means the presence of a nonnucleotide insertion. Oligonucleotide

Sequence

10-1

5′ CACGCCGCTG 3′

10-2

5′ CAGCGGCGTG 3′

10-1X

5′ [X]CACGCCGCTG 3′

10-2X

5′ [X]CAGCGGCGTG 3′

18-1

5′ GCATCACGCCGCTGAAGC 3′

18-2

5′ GCTTCAGCGGCGTGATGC 3′

17-1X

5′GCA[X]CACGCCGCTGAAGC 3′

17-2X

5′ GCT[X]CAGCGGCGTGATGC 3′


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Ammonia was removed in a CentriVap Vacuum Concentrator (Labconco, USA) at 15 °C, and the oligonucleotides were purified by reverse-phase (RP) HPLC on an Agilent 1200 series chromatograph using a Zorbax Eclipse XDB-C18 HPLC column 4.6 mm × 150 mm, (Agilent, USA) with a linear acetonitrile gradient (0 to 30%, v/v) in 0.02 M TEAA. Peak fractions were evaporated under reduced pressure, the bulk of triethylammonium acetate was removed by repeated coevaporation procedures with 50% (v/v) aqueous ethanol. The oligonucleotides were precipitated from aqueous solutions with a 10-fold volume of 2% LiClO4 in acetone (w/v). Upon centrifugation at 13000 × g for 3 min, the supernatants were discarded, the precipitate was washed twice with 1 mL of acetone and air dried. The DMTr group was next removed by treatment of all the oligonucleotides except 10-1X and 10-2X with 80% acetic acid for 5 min at ambient temperature, then the precipitation was repeated.

2.3. Synthesis of spin-labeled oligonucleotides Precipitated in the Li+ form, oligonucleotides (Scheme S3) 10-1X, 10-2X, 17-1X, and 172X were transferred to the cetyltrimethylammonium (CTA+) form, which is soluble in organic solvents. For this purpose, the oligonucleotides were dissolved in double-distilled and deionized water (ddH2O) at a concentration

> 150 OD260/mL (100 µL). A solution of

cetyltrimethylammonium bromide in water (25 µL, 8% cetyltrimethylammonium bromide) was added stepwise to the solutions. The precipitates of the CTA+ form of oligonucleotides were centrifuged, the supernatants were discarded, and the resulting precipitates were dried in vacuo over P2O5 for 24 h. The dried precipitates were dissolved in 80 µL of absolute dimethyl sulfoxide DMSO followed by addition of N,N-diisopropylethylamine (DIEA, 3 µL). A solution of TAMOSU in absolute DMSO (1.5 mg per 10 µL) was added, and the reaction mixtures were stirred at


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ambient temperature for 24 h. After that, the same amount of the TAM-OSU solution was added again, followed by stirring for another 24 h. Oligonucleotide components were precipitated by addition of the acetone solution (1 mL, 2% LiClO4) to the mixtures. The precipitates were washed with 1 mL of pure acetone, air dried, and dissolved in an aqueous NaOH solution (30 µL, 0.01 M) to hydrolyze the remaining succinimide moieties of the TAM residue. The solutions were stirred for 10 min, and the precipitation procedure was repeated. Oligonucleotides 10-1X and 10-2X were incubated with aqueous 80% acetic acid for 5 min at ambient temperature to cleave the DMTr group, precipitated with 2% LiClO4 in acetone, washed with pure acetone, and air dried. The dried precipitates were dissolved in ddH2O and purified by means of an acetonitrile gradient (0 to 30% for 30 min) in 0.02 M TEAA via reverse-phased (RP) HPLC. Peak fractions were collected, and the solvent was evaporated. The oligonucleotide derivatives were processed as described above and dissolved in ddH2O. ESI Mass spectra (ESI) were recorded. In average, the conversion to products containing oligonucleotide and label moieties in the ratio 1:1 was ~50% for the 10-nt oligonucleotides and ~25% for the 17-nt oligonucleotides (Figures S1 and S2). Purity of the obtained conjugates was checked by electrophoresis in a 20% denaturing polyacrylamide gel (PAAG).

2.4. Measurements of thermal stability and Circular Dichroism (CD) analyses of native and modified duplexes. The details are given is SI 2.5. Immobilization of a spin-labeled oligonucleotide using trehalose 60 µL of an aqueous solution of a TAM-labeled DNA complex in 10 mM sodium cacodylate (pH 7.0) containing 0.65 M trehalose was prepared by shock-freezing of the prepared


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solution in a 2-mL Eppendorf tube, then quickly transferred to a desiccator, followed by lyophilization in vacuum of 10−3 bar for 2 h at 193 K. The total concentration of each oligonucleotides for the sample was 2.4·10−5 M. Each sample was transferred into a quartz EPR tube (OD 3.8 mm, ID 2.8 mm for X-band) and dried by means of a turbomolecular pump (~5·10– 8

bar) for 24 hours to remove the remaining oxygen and water and to enhance relaxation time.35

2.6. Molecular dynamics (MD) simulations These simulations were performed using the AMBER 14 software.36 Structures of TAM with a terminal and internal non-nucleotide residue (Fig S5, S6) were generated in the xleap program (AmberTools 15), and particular atoms’ charges were calculated by the RESP method37 based on quantum mechanical calculation in the HF/6-31G*. Then, the library files were generated and applied for creation of “pdb” files containing the structure of the TAM-labeled DNA duplexes in the B-form. The MD simulations were performed in the parmbsc1 force field at explicit water shell.38 Twenty series of MD simulations with determined initial positions of spin labels and atoms’ speed distribution analysis were performed. Each series included 12 stages as described elsewhere.30

2.7. General EPR settings EPR experiments were carried out using commercial Bruker ELEXSYS E580 spectrometer equipped with ER 4118X-MD5W (X-band, 9 GHz) and EN5107D2 (Q-band, 34 GHz) resonators and Oxford Instruments temperature control system. The maximum available microwave power at Q-band was limited to 1 W.


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Samples with concentration about 2·10-5 M for Q-band measurements were prepared at room temperature in quartz capillary tubes (OD 1.65 mm, ID 1.15 mm, with the sample volume being ca. 10 µl), shock-frozen in liquid nitrogen and investigated at T=80 K. Room temperature DQC measurements at X-band were carried out using quartz tube (OD 3.8 mm, ID 2.8 mm) filled with 1 cm of trehalose powder. All pulse EPR experiments were carried out with repetition time of 300-400 µs and 3000 µs at 300 K and 80 K, respectively. The electron spin echo dephasing times (phase memory time), Tm, were measured by standard two-pulse echo sequence at field position corresponding to the maximum of the EPR spectrum. The pulse lengths were 10/20 ns (X-band) or 20/40 ns (Qband) for π/2 and π pulses, respectively. The DQC measurements were carried out at T=80 K (X-band) and 300 K (Q-band) by using six-pulse sequence, π/2 - τ1 - π - τ1 - π/2 - τ3 - π - τ3 - π/2 - τ2 - π -τ2 – echo14 with pulse lengths of 10/20 ns (X-band) or 20/40 ns (Q-band) for π/2 and π pulses, respectively. The DQC measurements were done at the field position corresponding to the maximum of the EPR spectrum. To filter out the dipolar modulation signal, the 64-step phase-cycling was applied. All obtained DQC traces were background corrected by third-order polynomial function and analyzed with Tikhonov regularization using DeerAnalysis program39. The DQC time trace at Q-band at 80 K was recorded by incrementing τ1 and decrementing τ2 in 16 ns steps. The initial values for τ1 were 5000 ns, the initial values for τ2 were 5400 ns, the delay τ3=50 ns remained constant. The total number of scans was 40. The number of shots per point was 10. Accumulation time was 5 h. The DQC time trace at X-band at 300 K was recorded by incrementing τ1 and decrementing τ2 in 16 ns steps. The initial values for τ1 were 2500 ns and 3000 ns for 10-1X-


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TAM/10-2X-TAM and 17-1X-TAM/17-2X-TAM correspondingly, the initial values for τ2 were 2800 ns and 3300 for 10-1X-TAM/10-2X-TAM and 17-1X-TAM/17-2X-TAM correspondingly, the delay τ3=30 ns remained constant. The total number of scans was 150 and 1000. The number of shots per point was 100. Accumulation time was 12 h and 150 h for 10-1X-TAM/10-2X-TAM and 17-1X-TAM/17-2X-TAM correspondingly.

3. RESULTS AND DISCUSSION In order to demonstrate and validate the new strategy for TAM-labeling of DNAs, we synthesized two pairs of complementary oligonucleotides. The first pair consists of 10 nt oligonucleotides with TAM labels attached at 5' terminal sites. The second pair involves 17 nt oligonucleotides with TAM labels introduced at non-nucleotide internal sites.

3.1. Preparation of spin-labeled DNA duplexes For the synthesis of the above double-stranded (ds) DNAs, first, the hydroxyl group of the previously obtained lactone (Scheme 1, structure 1) was dimetoxytritylated yielding the compound 2 (Scheme 1). Trans-1,4-diaminocyclohexane and piperazine were used as linkers for coupling with a reactive derivative of TAM label. After incorporation of the diamine (3), in order to carry out the following steps of automated oligonucleotide synthesis, it was necessary to protect the amino group by a trifluoroacetyl group (4). Phosphitylation, the final step in preparation of non-nucleoside monomer, yields phosphoramidite monomer with protected amino group of the side chain (5). Next, the synthesized monomer was subjected to an automated solidphase oligonucleotide synthesis (6). Standard protocols were used with the concentration of the monomer of 0.1 M and a longer condensation time of 15 min (see SI for details). As a result, we


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finally obtained two complementary 10 nt oligonucleotides bearing a non-nucleotide insertions at their terminal 5' positions, and two 17 nt oligonucleotides with internal non-nucleotide insertions (Scheme 1). In order to attach TAMs to non-nucleotide insertions, the as-synthesized oligonucleotides were fully deprotected in concentrated aqueous ammonia, purified by HPLC and treated with TAM-OSU as described previously.30 Finally, spin-labeled oligonucleotides were additionally purified by HPLC.

Scheme 1. Synthesis of the non-nucleoside phosphoramidite and its incorporation into DNA with further spin-labeling. As was mentioned above, we used two types of linkers to obtain 3 – trans-1,4diaminocyclohexane and piperazine (Scheme 1). However, in comparison with the primary amino group of trans-1,4-cyclohexanediamine, the secondary amino group of the piperazine moiety gave a spin-labeling product in a significantly smaller yield. We assume that the main reason for this is lower steric accessibility of piperazine residue compared to trans-1,4cyclohexanediamine. Because of that, we further proceeded only with the non-nucleoside monomer containing trans-1,4-diaminocyclohexane as a linker.


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The synthesized TAM-labeled oligonucleotides readily formed double stranded spinlabeled DNA duplexes 10-1X-TAM/10-2X-TAM and 17-1X-TAM/17-2X-TAM (Scheme 1). Prior to EPR distance measurements, we characterized structure and thermal stability of prepared dsDNAs. A circular dichroism (CD) study has shown that attachment of TAM label using nonnucleotide insertion at 5' ends of oligonucleotides does not severely perturb the B-form conformation of dsDNA (SI). However, the incorporation of non-nucleotide insertion into the internal positions of oligonucleotides 17-1X and 17-2X slightly decreases the amplitude of CD signal of doubly spin-labeled DNA complex compared to similar native 18 base pair (bp) duplex (Table S4, complex IV). This is possibly caused by a smaller number of nucleotides, weakening of base-pairing and stacking interactions in the spin-labeled duplex (SI). The CD data are consistent with the results of thermal stability measurements on spinlabeled complexes. The attachment of non-nucleotide insertions and TAM-residues at 5' ends of DNA duplex (Table S4, complexes II and III) does not significantly change the stability of 10 bp duplexes. However, in the case of 17 bp duplexes the presence of non-nucleotide insertion destabilizes the DNA complexes (Table S4, complexes V and VI). The thermodynamic changes ° ° (∆∆S° = ∆S௠௢ௗ௜௙௜௘ௗ − ∆S௡௔௧௜௩௘ ) of one insertion give the absolute values of ∆∆S௑° = 54.5

cal⋅M-1⋅K-1 and ∆∆H௑° = 20.5 kcal⋅M-1, which are consistent with previously published data.40 The presence of two internal insertions (Table S4, complexes V and VI) within the structure of the DNA complex leads to increase of the thermodynamic changes by more than a factor 2, ° ° resulting in the absolute values of ∆∆S௑௑ and ∆∆H௑௑ parameters of complex VII to 165.5

cal⋅M-1⋅K-1 and 60.7 kcal⋅M-1, correspondingly. The insertion of TAM labels into the structure of 17 nt oligonucleotides containing non-nucleotide insertion additionally decreases the thermal stability of DNA complex (Table S4, complex VIII). Note that for longer DNAs and their


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complexes, more relevant for modeling of natural biosystems, similar attachment of TAMs at internal sites should have much weaker effect on thermal stability.

3.2. Spin-spin distance measurements in TAM-labeled DNA duplexes To measure the interspin distances between two TAM labels in DNA duplexes, we employed X/Q-band DQC at 80 and 298 K. In order to increase the phase memory time (Tm) experiments were carried out in deuterated solvents. Figure 1 shows DQC time traces, corresponding distance distributions and MD simulations obtained for 10-1X-TAM/10-2X-TAM and 17-1X-TAM/17-2X-TAM duplexes in frozen water/glycerol solution at 80 K and in glassy trehalose at 298 K. The mean distances and distribution widths (defined as the standard deviation parameter σ) are summarized in Table 2. The width of the distance distribution obtained at 80 K for 10-1X-TAM/10-2X-TAM (σ =0.5 nm) is larger than those obtained previously for 10 bp DNA duplexes 5' TAM-labeled via piperazine linker (σ=0.2 nm).30,33 The width increases to the larger extent when TAM labels are introduced at internal positions of 17-1X-TAM/17-2X-TAM duplexes (σ =0.9 nm). Our previous studies have shown that the application of TAM spin-labels in combination with immobilization in trehalose provides the longest Tm values known up to date at room temperature for spin-labeled biopolymers. Therefore, in this work we also used trehalose as an immobilizer for distance measurements at 298 K. In order to prevent the melting of DNA we used the strategy of sample preparation published previously.35 The key step of this method is lyophilization of the water/trehalose sample from a frozen state in vacuum immediately after shock-freezing in liquid nitrogen. In order to minimize the contribution to 1/Tm from ganisotropy, the distance measurements at 298 K were performed at X-band.23 It has been found


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that 10-1X-TAM/10-2X-TAM and 17-1x-TAM/17-2x-TAM duplexes immobilized in dry trehalose have the same Tm=2 µs at 298 K, which is very close to those obtained previously for TAM-labeled DNA.35 Table 2. Mean distances and distribution width parameters σ obtained by EPR for all studied complexes. Duplex

Immobilizer, (Temperature, K)

± σ/ nm

10-1X-TAM/10-2X-TAM

Water/glycerol, (80 K)

5.0±0.5

10-1X-TAM/10-2X-TAM

Trehalose, (298 K)

5.0±0.7

17-1X-TAM/17-2X-TAM

Water/glycerol, (80 K)

5.7±0.9

17-1X-TAM/17-2X-TAM

Trehalose, (298 K)

5.4±0.7

DQC time traces in glassy trehalose at 298 K were measured within a shorter span (4 and 5 µs) than at 80 K (9.5 µs) due to the lower sensitivity. Because of that only and σ values, but not the shape of the distribution, can be analyzed in the range >4 nm at 298 K. The and σ values for 10-1X-TAM/10-2X-TAM obtained at 298 K in trehalose agree well with those measured at 80 K in water/glycerol (Table 2). In case of 17-1x-TAM/17-2x-TAM, and σ values at 298 K are slightly smaller than at 80 K (Table 2). Such decrease is attributed to the shorter dipolar evolution time at 298 K, which limits the largest available distance to ~7 nm. In general, there is a good agreement between distance distributions at room temperature and those obtained in water/glycerol solution at 80 K. Thus, we emphasize the applicability of the new spin-labelling strategy for structural EPR studies at ambient and physiological temperatures. Previous studies have demonstrated that hydrophobic interaction of TAM radicals with terminal base pairs of the DNA duplex fosters TAMs to occupy well-defined “capping”


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positions.41 When semi-rigid piperazine linker is used, this results in smaller conformational flexibility of TAM label and a narrower distance distribution.33 To elucidate similar effects for duplexes with non-nucleotide insertions, we performed a series of molecular dynamics (MD) simulations.

Figure 1. DQC distance measurements in duplexes 10-1X-TAM/10-2X-TAM (A) and 17-1XTAM/17-2X-TAM (B). Panels (I-II) show Q-band data at 80 K in water/glycerol (red lines), Xband data at 298 K in glassy trehalose (blue lines), and MD simulations (green lines). All experimental traces were background corrected and normalized. Black lines show best fits of time-domain data obtained by Tikhonov regularization using DeerAnalysis2013.39 Typical conformations of TAM-labeled duplexes are shown in panels (III).


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MD simulations for 10-1X-TAM/10-2X-TAM and 17-1X-TAM/17-2X-TAM duplexes were carried out in an explicit solvent at 298 K in an NPT ensemble for 2 µs using the AMBER14 software.36 The introduction of non-nucleotide insertions with TAM labels does not change the structure of 10-1X-TAM/10-2X-TAM DNA duplex: root-mean-square deviation (RMSD) from the native (unlabeled) structure is 0.6 Å. The interspin distance averaged over the trajectory equals to 4.90 nm with RMSD 0.52 nm (SI). The maximum of the distribution at 4.58 nm corresponds to the “capped” structure. Another peak with the maximum at 5.01 nm corresponds to the structure where the TAM labels do not interact with the DNA duplex. Calculated distribution is in excellent agreement with that measured experimentally by DQC. The MD analysis of 17-1X-TAM/17-2X-TAM complexes shows a number of conformations for each non-nucleotide insertion with TAM. It can interact with the terminal base pair of the duplex or freely move without interaction with DNA (SI). These lead to the significant broadening of the interspin distance distribution and average distance of 5.56 ± 0.86 nm. Thus, the attachment of TAM labels at internal positions of complex 17-1X-TAM/17-2XTAM causes the broadening of interspin distribution according to both experimental DQC and calculated MD data. The main reason for this is the high conformational flexibility of nonnucleotide insertion. Possible “capped” conformation of TAM labels, which was observed for the 10-1X-TAM/10-2X-TAM, is not feasible in the case of internal incorporation of TAM label in 171X-TAM/17-2X-TAM complex. Therefore, the broadening of interspin distance distribution in case of non-nucleotide insertion compared to previous TAM-labeling at the DNA termini30 is caused by larger length of the linker and by its higher conformational flexibility.


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4. CONCLUSIONS In this work we for the first time proposed a convenient approach for site-directed spin labeling of DNAs with TAM radicals. While the previous TAM-labeling scheme was applicable only for the label attachment at DNA termini, the present one is more versatile and allows introduction of TAMs at either terminal or internal sites of the DNAs. This drastically broadens the scope of applications of such method. This method even overcomes some limitations known for nitroxide-based spin labeling, such as possible formation of diastereomers in case of internucleotide phosphorothioate labeling42,43 and possible alteration of Watson-Crick base pairing when the label is attached to the base.44–47 The new method can also be applied for sitedirected RNA labeling as an alternative to such complicated approaches as, e.g., complementaryaddressed SDSL.48,49 We foresee that many structures, such as DNA quadruplexes, kissing loops and complexes with proteins can now become available for structural EPR studies using TAMs. Most importantly, these studies can be conducted at room temperature using the existing immobilization approaches, e.g. in glassy trehalose. We need to seek further ways to reduce the linker length and flexibility, in order to enhance the accuracy of nanoscale distance measurements. However, even as is now, the proposed approach can be broadly used, especially in cases where the conformational changes in biopolymers are suspected at cryogenic temperatures. In such cases the opportunity of physiological-temperature distance measurements using the developed TAM-based strategy will be greatly appreciated as a new independent and informative tool.


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ASSOCIATED CONTENT Supporting Information General procedures and characterization methods. Synthesis of TAM-hydroxysuccinimide derivative. Synthesis of non-nucleoside phosphoramidite monomer for automated solid-phase oligonucleotide synthesis. Oligonucleotide synthesis. Synthesis of spin-labeled oligonucleotides. Quantification of spin-labeled oligonucleotides. Thermal stability data of native and modified complexes. Circular dichroism analyses. Immobilization of spin-labelled oligonucleotide using trehalose. Molecular dynamics simulation. General EPR settings. Phase memory time measurements. Raw DQC data. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * (D.V.P.) E-mail: [email protected]. * (E.G.B.) E-mail: [email protected]. * (M.V.F.) E-mail: [email protected]. Notes The authors declare no competing financial interest ACKNOWLEDGMENT D.V.P., M.S.K. would like to acknowledge RFBR grant (No. 16-04-01029 A) and the ICBFM SB RAS budget project (VI.62.1.4, 0309-2016-0004) for the financial support for the synthesis


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of non-nucleoside monomer. V.M.T. would like to acknowledge the NIOC SB RAS budget project (0302-2016-0002). E.G.B. and other thanks RSF (No. 14-14-00922). REFERENCES (1)

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Scheme 1. Synthesis of the non-nucleoside phosphoramidite and its incorporation into DNA with further spin-labeling. 254x190mm (96 x 96 DPI)


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Figure 1. DQC distance measurements in duplexes 10-1x-TAM/10-2x-TAM (A) and 17-1x-TAM/17-2x-TAM (B). Panels (I-II) show Q-band data at 80 K in water/glycerol (red lines), X-band data at 298 K in trehalose (blue lines), and MD simulations (green lines). All experimental traces were background corrected and normalized. Black lines show best fits of time-domain data obtained by Tikhonov regularization using DeerAnalysis2013.[39] Typical conformations of TAM-labeled duplexes are shown in panels (III). 76x108mm (300 x 300 DPI)



TOC 41x28mm (300 x 300 DPI)


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A Versatile Approach to Attachment of Triarylmethyl Labels to DNA for

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